The antecedents, attributes and consequences of trust among nurses and nurse managers: a concept analysis.

BACKGROUND: Although trust has been investigated in the health context, limited research explores nurse and nurse manager perceptions of trust. OBJECTIVE: To explore the concept of trust amongst nurses and nurse managers at individual, interpersonal and organisational levels. DESIGN: Our paper reports the findings from an interpretivist study conducted within the British National Health Service, involving thirty-nine semi-structured interviews with nurses and nurse managers. SETTINGS: Large acute and small community organisation within the British National Health Service. PARTICIPANTS: 28 nurses and 11 nurse managers working within an Acute and a Community sector organisation - 20 and 19 in each organisation. Participants were selected through a process of purposive sampling, reflecting variations in terms of age, grade, ward and tenure. METHODS: We utilise a concept analysis framework in exploring the antecedents, attributes and consequences of trust amongst nurses and nurse managers at individual, interpersonal and organisational levels. RESULTS: Key findings suggest that trust is formed within the immediate ward environment, and is significantly influenced by the role of line manager. Other positively influencing factors include professionalism and commitment to the nursing profession. These form the basis for the teamwork, delegation, support, open communication systems, confidentiality and discretion essential to delivering quality patient care. Negatively influencing factors include new management concepts, practices and styles overseen by managers recruited from the private sector. New management concepts were associated with reductions in the number of qualified nurses and increasing numbers of untrained nursing staff, reduced direct patient contact, less opportunities for professional training and development and deteriorating terms and conditions of employment. CONCLUSIONS: Our findings offer insight for managers, nurses and human resource practitioners to help build high trust relationships in a health care context. Of particular import is the need for managers to communicate more effectively organisational and financial constraints, in a manner that does not 'alienate' nurses and nurse managers, by highlighting their value and acknowledging their role in delivering high quality patient care.

Psychological contracts and commitment amongst nurses and nurse managers: a discourse analysis.

BACKGROUND: Few studies explore the link between the psychological contracts and the commitment of nursing professionals in the healthcare sector, and how perceived breaches of the psychological contract can impact on nurses' commitment levels. OBJECTIVE: This study explores the connections between the psychological contracts and organisational and professional commitment of nurses and nurse managers. DESIGN: Semi-structured interviews were conducted with nurses and nurse managers, to explore the connections between their psychological contracts and organisational and professional commitment. SETTINGS: Large acute and small community organisation within the British National Health Service. PARTICIPANTS: 28 nurses and 11 nurse managers working within an acute and a community sector organisation - 20 and 19 in each organisation. Participants were selected through a process of purposive sampling, reflecting variations in terms of age, grade, ward and tenure. METHODS: A discourse analysis was conducted on the qualitative data from the thirty nine semi-structured interviews. RESULTS: Two overall themes emerged, professional and managerial values. Professional values included the sub-themes: professional recognition; immediate work environment - leadership and peer support; professional development and progression. Sub-themes under managerial values included: involvement; general management; resource management. CONCLUSIONS: The findings suggest that nurses and nurse managers are governed by relational psychological contracts, underpinned by an affective and to a lesser extent normative commitment towards the nursing profession. They emphasise 'professional values', and professional commitment, as the basis for positive psychological contracts amongst nursing professionals. There was anecdotal evidence of relational psychological contract breach, with decreasing job satisfaction as the outcome of perceived psychological contract breach. Positive psychological contracts and commitment levels amongst nursing professionals can be supported by managers been aware and sensitive to nursing discourses, and managing their expectations through greater involvement and leadership development.

Immunisation: the views of parents and health professionals in Ireland.

A knowledge, attitude and practice study on vaccinations was undertaken among Irish parents and healthcare professionals between May and August 2001. Parents expressed fear of vaccine side effects, mistrust of health services, and felt poorly informed on the vaccination issues. According to group discussions, health professionals felt they lack time and user-friendly materials to properly inform the parents.

Induction of rabbit lung CYP4A4 prostaglandin omega-hydroxylase by various steroid hormones.

Cytochrome P4504A4 (CYP4A4) is expressed at low basal levels in adult rabbit lungs, but is significantly induced during pregnancy by an unknown mechanism. As the gradual rise in CYP4A4 levels appears to coincide with the progressive increase in several steroid hormones throughout pregnancy, we examined the induction of CYP4A4 after treatment with various steroid hormones by monitoring both the CYP4A4 mRNA level and the CYP4A4-specific prostaglandin E(1) (PGE(1)) omega-hydroxylation reaction in rabbit lung microsomes. Treatment with progesterone and/or a synthetic glucocorticoid (dexamethasone) resulted in a significant increase in PGE(1) omega-hydroxylase activity, whereas estradiol, aldosterone, dehydroepiandrosterone, and dehydroepiandrosterone sulfate did not. These studies indicated that dexamethasone was a more potent inducer of CYP4A4 than progesterone. Simultaneous injection of dexamethasone and glucocorticoid/progesterone antagonists (RU38486, RU40555, or RU43044) inhibited the increase in PGE(1) omega-hydroxylase activity as well as mRNA levels by approximately 50%. In addition, simultaneous treatment with both dexamethasone and progesterone did not result in an additive or synergistic effect on PGE(1) omega-hydroxylase activity. These data indicate that, while distinctive receptors for glucocorticoid and/or progesterone are involved, induction may also require common or interacting regulatory elements (yet to be determined) in the CYP4A4 gene. These findings implicate both of these steroid receptors (PR/GR) in the induction of CYP4A4 in rabbit lung.

Acidic hydrolysis as a mechanism for the cleavage of the Glu(298)-->Asp variant of human endothelial nitric-oxide synthase.

The 894G-->T polymorphism within exon 7 of the human endothelial nitric-oxide synthase (eNOS) gene codes for glutamate or aspartate, respectively, at residue 298 and has been associated with several diseases of cardiovascular origin. A recent report indicates that Asp(298)-eNOS (E298D) is cleaved intracellularly to 100- and 35-kDa fragments, suggesting a mechanism for reduced endothelial function. Here we have documented the precise cleavage site of the E298D variant as a unique aspartyl-prolyl (Asp(298)--Pro(299)) bond not seen in wild-type eNOS (Glu(298)). We show that E298D-eNOS, as isolated from cells and in vitro, is susceptible to acidic hydrolysis, and the 100-kDa fragment can be generated ex vivo by increasing temperature at low pH. Importantly, cleavage of E298D was eliminated using a sample buffer system designed to limit acidic hydrolysis of Asp--Pro bonds. These results argue against intracellular processing of E298D-eNOS and suggest that previously described fragmentation of E298D could be a product of sample preparation. We also found that eNOS turnover, NO production, and the susceptibility to cellular stress were not different in cells expressing WT versus E298D-eNOS. Finally, enzyme activities were identical for the respective recombinant enzymes. Thus, intracellular cleavage mechanisms are unlikely to account for associations between the exon 7 polymorphism and cardiovascular diseases.

Direct interaction between endothelial nitric-oxide synthase and dynamin-2. Implications for nitric-oxide synthase function.

Endothelial nitric-oxide synthase (eNOS) is regulated in part through specific protein interactions. Dynamin-2 is a large GTPase residing within similar membrane compartments as eNOS. Here we show that dynamin-2 binds directly with eNOS thereby augmenting eNOS activity. Double label confocal immunofluorescence demonstrates colocalization of eNOS and dynamin in both Clone 9 cells cotransfected with green fluorescent protein-dynamin and eNOS, as well as in bovine aortic endothelial cells (BAEC) expressing both proteins endogenously, predominantly in a Golgi membrane distribution. Immunoprecipitation of eNOS from BAEC lysate coprecipitates dynamin and, conversely, immunoprecipitation of dynamin coprecipitates eNOS. Additionally, the calcium ionophore, a reagent that promotes nitric oxide release, enhances coprecipitation of dynamin with eNOS in BAEC, suggesting the interaction between the proteins can be regulated by intracellular signals. In vitro studies demonstrate that glutathione S-transferase (GST)-dynamin-2 quantitatively precipitates both purified recombinant eNOS protein as well as in vitro transcribed (35)S-labeled eNOS from solution indicating a direct interaction between the proteins in vitro. Scatchard analysis of binding studies demonstrates an equilibrium dissociation constant (K(d)) of 27.6 nm. Incubation of purified recombinant eNOS protein with GST-dynamin-2 significantly increases eNOS activity as does overexpression of dynamin-2 in ECV 304 cells stably transfected with eNOS-green fluorescent protein. These studies demonstrate a direct protein-protein interaction between eNOS and dynamin-2, thereby identifying a new NOS-associated protein and providing a novel function for dynamin. These events may have relevance for eNOS regulation and trafficking within vascular endothelium.

Reconstitution of an endothelial nitric-oxide synthase (eNOS), hsp90, and caveolin-1 complex in vitro. Evidence that hsp90 facilitates calmodulin stimulated displacement of eNOS from caveolin-1.

The activity of endothelial nitric-oxide synthase (eNOS) is regulated by its subcellular localization, phosphorylation and through its interaction with different proteins. The association of eNOS with caveolin-1 (Cav) is believed to maintain eNOS in an inactive state; however, increased association of eNOS to heat shock protein 90 (hsp90) is observed following activation. In this study, we investigate the relationship between caveolin and hsp90 as opposing regulatory proteins on eNOS function. Immunoprecipitation of Cav-1 from bovine lung microvascular endothelial cells shows that eNOS and hsp90 are present in the Cav-1 complex. eNOS and hsp90 from the lysate also interact with exogenous glutathione S-transferase-linked caveolin-1 (GST-Cav), and the addition of calcium-activated calmodulin (CaM) to the GST-Cav complex partially inhibited the association of eNOS and hsp90. Purified eNOS associates with GST-Cav specifically through the caveolin-scaffolding domain (residues 82-101); however, the addition of CaM slightly, but nonstatistically, reduces eNOS binding to GST-Cav. When hsp90 is present in the binding reaction, the addition of increasing concentrations of CaM significantly displaces eNOS and hsp90 from GST-Cav. eNOS enzymatic activity is also less sensitive to inhibition by the caveolin scaffolding peptide (residues 82-101) when eNOS is prebound to hsp90. Collectively, our results show that the actions of CaM on eNOS dissociation from caveolin are facilitated in the presence of hsp90.

Enhanced electron flux and reduced calmodulin dissociation may explain "calcium-independent" eNOS activation by phosphorylation.

Bovine endothelial nitric oxide synthase (eNOS) is phosphorylated directly by the protein kinase Akt at serine 1179. Mutation of this residue to the negatively charged aspartate (S1179D eNOS) increases nitric oxide (NO) production constitutively, in the absence of agonist challenge. Here, we examine the potential mechanism of how aspartate at 1179 increases eNOS activity using purified proteins. Examination of NO production and cytochrome c reduction resulted in no substantial changes in the K(m)/EC(50) for L-arginine, calmodulin, and calcium, whereas there was a 2-fold increase in the rate of NO production for S1179D and a 2-4-fold increase in reductase activity (based on cytochrome c reduction). The observed increase in activity for both assays of NOS function indicates that a faster rate of electron flux through the reductase domain is likely the rate-limiting step in NO formation from eNOS. In addition, S1179D eNOS did show an increased resistance to inactivation by EGTA compared with wild type eNOS. These results suggest that a negative charge imposed at serine 1179, either by phosphorylation or by replacement with aspartate, increases eNOS catalytic activity by increasing electron flux at the reductase domain and by reducing calmodulin dissociation from activated eNOS when calcium levels are low.

Regulation of endothelium-derived nitric oxide production by the protein kinase Akt.

Endothelial nitric oxide synthase (eNOS) is the nitric oxide synthase isoform responsible for maintaining systemic blood pressure, vascular remodelling and angiogenesis. eNOS is phosphorylated in response to various forms of cellular stimulation, but the role of phosphorylation in the regulation of nitric oxide (NO) production and the kinase(s) responsible are not known. Here we show that the serine/threonine protein kinase Akt (protein kinase B) can directly phosphorylate eNOS on serine 1179 and activate the enzyme, leading to NO production, whereas mutant eNOS (S1179A) is resistant to phosphorylation and activation by Akt. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and activation-deficient Akt attenuates NO production stimulated by vascular endothelial growth factor. Thus, eNOS is a newly described Akt substrate linking signal transduction by Akt to the release of the gaseous second messenger NO.

Sparing of mdx extraocular muscles from dystrophic pathology is not attributable to normalized concentration or distribution of neuronal nitric oxide synthase.

Previous findings have led to speculations that decreased concentration of nNOS (neuronal nitric oxide synthase) may underlie some aspects of the pathophysiology of dystrophic muscle. We have tested whether the sparing of extraocular muscles (EOM) in muscular dystrophy is attributable to the presence of normal nNOS concentration and distribution in these muscles. Measurements of total nNOS concentration in control muscle showed that total nNOS comprises approximately 0.05% of total muscle protein, indicating a molar stoichiometry of approximately 60 and 20 to total dystrophin and syntrophin, respectively. Thus, most muscle nNOS is either not associated with the dystrophin complex, or binds to yet unidentified sites in the complex. nNOS concentration was at least two-fold greater in C57 EOM and tibialis anterior (TA) compared with mdx samples. No significant differences in nNOS concentration in EOM versus TA in either mdx or C57 mice were observed, nNOS was concentrated at the sarcolemma of all C57 samples, while mdx nNOS displayed a cytosolic distribution, except in fibers that reverted to express dystrophin. These data show that mdx EOM are spared by a mechanism other than normalized concentration and location of nNOS.

Relationships between NADPH diaphorase staining and neuronal, endothelial, and inducible nitric oxide synthase and cytochrome P450 reductase immunoreactivities in guinea-pig tissues.

The presence of NADPH diaphorase staining was compared with the immunohistochemical localization of four NADPH-dependent enzymes-neuronal (type I), inducible (type II), and endothelial (type III) nitric oxide synthase (NOS) and cytochrome P450 reductase. Cell types that were immunoreactive for the NADPH-dependent enzymes were also stained for NADPH diaphorase, suggesting that endothelial and neuronal NOS and cytochrome P450 reductase all show NADPH diaphorase activity in formaldehyde-fixed tissue. However, in some tissues, the presence of NADPH diaphorase staining did not coincide with the presence of any of the NADPH-dependent enzymes we examined. In vascular endothelial cells, the punctate pattern of staining observed with NADPH diaphorase histochemistry was identical to that seen following immunohistochemistry using antibodies to endothelial NOS. In enteric and pancreatic neurons and in skeletal muscle, the presence of NADPH diaphorase staining correlated with the presence of neuronal NOS. In the liver, sebaceous glands of the skin, ciliated epithelium, and a subpopulation of the cells in the subserosal glands of the trachea, zona glomerulosa of the adrenal cortex, and epithelial cells of the lacrimal and salivary glands, the presence of NADPH diaphorase staining coincided with the presence of cytochrome P450 reductase immunoreactivity. In epithelial cells of the renal tubules and zona fasciculata and zona reticularis of the adrenal cortex, NADPH diaphorase staining was observed that did not coincide with the presence of any of the enzymes. Inducible NOS was not observed in any tissue. Thus, while tissues that demonstrate immunoreactivity for neuronal and endothelial NOS also stain positively for NADPH diaphorase activity, the presence of NADPH diaphorase staining does not reliably or specifically indicate the presence of one or more NOS isoforms.

Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy.

Neuronal nitric oxide synthase (nNOS) in fast-twitch skeletal muscle fibers is primarily particulate in contrast to its greater solubility in brain. Immunohistochemistry shows nNOS localized to the sarcolemma, with enrichment at force transmitting sites, the myotendinous junctions, and costameres. Because this distribution is similar to dystrophin, we determined if nNOS expression was affected by the loss of dystrophin. Significant nNOS immunoreactivity and enzyme activity was absent in skeletal muscle tissues from patients with Duchenne muscular dystrophy. Similarly, in dystrophin-deficient skeletal muscles from mdx mice both soluble and particulate nNOS was greatly reduced compared with C57 control mice. nNOS mRNA was also reduced in mdx muscle in contrast to mRNA levels for a dystrophin binding protein, alpha 1-syntrophin. nNOS levels increased dramatically from 2 to 52 weeks of age in C57 skeletal muscle, which may indicate a physiological role for NO in aging-related processes. Biochemical purification readily dissociates nNOS from the dystrophin-glycoprotein complex. Thus, nNOS is not an integral component of the dystrophin-glycoprotein complex and is not simply another dystrophin-associated protein since the expression of both nNOS mRNA and protein is affected by dystrophin expression.

Melatonin administration prevents lipopolysaccharide-induced oxidative damage in phenobarbital-treated animals.

The protective effect of melatonin on lipopolysaccharide (LPS)-induced oxidative damage in phenobarbital-treated rats was measured using the following parameters: changes in total glutathione (tGSH) concentration, levels of oxidized glutathione (GSSG), the activity of the antioxidant enzyme glutathione peroxidase (GSH-PX) in both brain and liver, and the content of cytochrome P450 reductase in liver. Melatonin was injected intraperitoneally (ip, 4mg/kg BW) every hour for 4 h after LPS administration; control animals received 4 injections of diluent. LPS was given (ip, 4 mg/kg) 6 h before the animals were killed. Prior to the LPS injection, animals were pretreated with phenobarbital (PB), a stimulator of cytochrome P450 reductase, at a dose 80 mg/kg BW ip for 3 consecutive days. One group of animals received LPS together with Nw-nitro-L-arginine methyl ester (L-NAME), a blocker of nitric oxide synthase (NOS) (for 4 days given in drinking water at a concentration of 50 mM). In liver, PB, in all groups, increased significantly both the concentration of tGSH and the activity of GSH-PX. When the animals were injected with LPS the levels of tGSH and GSSG were significantly higher compared with other groups while melatonin and L-NAME significantly enhanced tGSH when compared with that in the LPS-treated rats. Melatonin alone reduced GSSG levels and enhanced the activity of GSH-PX in LPS-treated animals. Additionally, LPS diminished the content of cytochrome P450 reductase with this effect being largely prevented by L-NAME administration. Melatonin did not change the content of P450 either in PB- or LPS-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional aspects of eicosanoid hydroxylation by lung and kidney cytochromes P450. Expression of cDNAs in mammalian cells and E. coli.

A gene subfamily of cytochromes P450 with catalytic activity toward various eicosanoid substrates has been studied with a variety of techniques in this laboratory, including purification and characterization, localization at the tissue and subcellular levels, physiological function, and cloning and expression in prokaryotic and eukaryotic systems. This paper reports experiments directed toward determining the function of the cytochrome P4504A metabolite, 20-hydroxyarachidonic acid (20-hydroxyeicosatetraenoic acid; 20-HETE), in cellular ion flux, immunohistochemical localization in lung, the effects of a mechanism-based inhibitor, 12-hydroxy-16-heptadecynoic acid (12-HHDYA) on PGE1 omega-hydroxylation, and the structure-function determinants which govern the activities of the enzymes encoded by this gene subfamily.

31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase.

31P NMR spectroscopy has been utilized in conjunction with site-directed mutagenesis and phospholipid analysis to determine structural aspects of the prosthetic flavins, FAD and FMN, of NADPH-cytochrome P450 reductase. Comparisons are made among detergent-solubilized and protease (steapsin)-solubilized preparations of porcine liver reductases, showing unequivocally that the 31P NMR signals at approximately 0.0 ppm in the detergent-solubilized, hydrophobic form are attributable to phospholipids. By extraction and TLC analysis, the phospholipid contents of detergent-solubilized rat liver reductase, both tissue-purified and Escherichia coli-expressed, have been determined to reflect the membranes from which the enzyme was extracted. In addition, the cloned, wild-type NADPH-cytochrome P450 reductase exhibits an additional pair of signals downfield of the normal FAD pyrophosphate resonances reported by Otvos et al. [(1986) Biochemistry 25, 7220-7228], but these signals are not observed with tissue-purified or mutant enzyme preparations. The Tyr140----Asp140 mutant, which exhibits only 20% of wild-type activity, displays no gross changes in 31P NMR spectra. However, the Tyr178----Asp178 mutant, which has no catalytic activity and does not bind FMN, exhibits no FMN 31P NMR signal and a normal, but low intensity, pair of signals for FAD. The latter experiments, taking advantage of mutations in residues putatively on either side of the FMN isoalloxazine ring, suggest subtle to severe changes in the binding of the flavin prosthetic groups and, perhaps, cooperative interactions of flavin binding to NADPH-cytochrome P450 reductase.